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Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol

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ABSTRACT

Background: Opioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus.

Methods: Neonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2–6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons.

Results: In the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol’s ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons.

Conclusions: These results suggest that ethanol’s neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol.

Electronic supplementary material: The online version of this article (doi:10.1186/s12974-017-0844-3) contains supplementary material, which is available to authorized users.

No MeSH data available.


In vitro effects of ethanol with or without opioid receptor agonists on cytokine secretion from hypothalamic microglia in primary cultures. Showing the changes in the protein levels of pro-inflammatory cytokines TNF-α (a), IFN-γ (b), IL-1α (c), IL-1β (d), IL-6 (e), MIP-3α (f), MCP-1 (g), M-CSF (h), CXCL1 (i), and RANTES (j) and anti-inflammatory cytokines IL-4 (k) and IL-13 (l) in microglial conditioned medium following 24-h treatment with ethanol (50 mM) with or without MOR agonist (DAMGO, 50 μM) or DOR agonist (DPDPE, 10 nM). Data are represented as mean ± SEM (n = 6). Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between control and other treatment groups or ethanol and ethanol and opioidergic drug groups are shown by lines with p values on the top of bar graphs
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Fig5: In vitro effects of ethanol with or without opioid receptor agonists on cytokine secretion from hypothalamic microglia in primary cultures. Showing the changes in the protein levels of pro-inflammatory cytokines TNF-α (a), IFN-γ (b), IL-1α (c), IL-1β (d), IL-6 (e), MIP-3α (f), MCP-1 (g), M-CSF (h), CXCL1 (i), and RANTES (j) and anti-inflammatory cytokines IL-4 (k) and IL-13 (l) in microglial conditioned medium following 24-h treatment with ethanol (50 mM) with or without MOR agonist (DAMGO, 50 μM) or DOR agonist (DPDPE, 10 nM). Data are represented as mean ± SEM (n = 6). Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between control and other treatment groups or ethanol and ethanol and opioidergic drug groups are shown by lines with p values on the top of bar graphs

Mentions: We employed multiplex ELISA and primary cultures of hypothalamic microglia in order to characterize secretion patterns of all major cytokines by microglia following an ethanol challenge in the presence and absence of opioid receptor agonists. In the initial dose-response study employing various doses of ethanol (25–100 mM), we identified that a 50 mM dose of ethanol maximally increased the levels of ten pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1α, IL-1β, IL-6, MIP-3α, MCP-1, M-CSF, CXCL1, chemokine (C-C motif) ligand 5 (RANTES)) and reduced two anti-inflammatory cytokines (IL-4 and IL-13) as compared to the control group (Fig. 4). The cytokine responses to 50 mM dose of ethanol were compared in the presence and absence of MOR or DOR agonists (Fig. 5). Like ethanol, the MOR agonist DAMGO increased the levels of all ten pro-inflammatory cytokines and reduced the two anti-inflammatory cytokines. When combined, DAMGO was not able to change the levels of most of the pro-inflammatory and anti-inflammatory cytokines induced by ethanol, with exception of MIP-3α which was moderately elevated (Fig. 5f) and RANTES, which was moderately decreased (Fig. 5j). The DOR agonist DPDPE alone did not change the basal levels of pro-inflammatory cytokines (Fig. 5a–j) but increased the basal levels of anti-inflammatory cytokines (Fig. 5k, l). DPDPE also prevented ethanol’s stimulatory actions on pro-inflammatory cytokines and ethanol’s inhibitory actions on anti-inflammatory cytokines. These results support the concept that MOR and DOR function differently in hypothalamic microglia and identify a pro-inflammatory function for MOR and anti-inflammatory function for DOR.Fig. 4


Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol
In vitro effects of ethanol with or without opioid receptor agonists on cytokine secretion from hypothalamic microglia in primary cultures. Showing the changes in the protein levels of pro-inflammatory cytokines TNF-α (a), IFN-γ (b), IL-1α (c), IL-1β (d), IL-6 (e), MIP-3α (f), MCP-1 (g), M-CSF (h), CXCL1 (i), and RANTES (j) and anti-inflammatory cytokines IL-4 (k) and IL-13 (l) in microglial conditioned medium following 24-h treatment with ethanol (50 mM) with or without MOR agonist (DAMGO, 50 μM) or DOR agonist (DPDPE, 10 nM). Data are represented as mean ± SEM (n = 6). Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between control and other treatment groups or ethanol and ethanol and opioidergic drug groups are shown by lines with p values on the top of bar graphs
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Related In: Results  -  Collection

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Fig5: In vitro effects of ethanol with or without opioid receptor agonists on cytokine secretion from hypothalamic microglia in primary cultures. Showing the changes in the protein levels of pro-inflammatory cytokines TNF-α (a), IFN-γ (b), IL-1α (c), IL-1β (d), IL-6 (e), MIP-3α (f), MCP-1 (g), M-CSF (h), CXCL1 (i), and RANTES (j) and anti-inflammatory cytokines IL-4 (k) and IL-13 (l) in microglial conditioned medium following 24-h treatment with ethanol (50 mM) with or without MOR agonist (DAMGO, 50 μM) or DOR agonist (DPDPE, 10 nM). Data are represented as mean ± SEM (n = 6). Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between control and other treatment groups or ethanol and ethanol and opioidergic drug groups are shown by lines with p values on the top of bar graphs
Mentions: We employed multiplex ELISA and primary cultures of hypothalamic microglia in order to characterize secretion patterns of all major cytokines by microglia following an ethanol challenge in the presence and absence of opioid receptor agonists. In the initial dose-response study employing various doses of ethanol (25–100 mM), we identified that a 50 mM dose of ethanol maximally increased the levels of ten pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1α, IL-1β, IL-6, MIP-3α, MCP-1, M-CSF, CXCL1, chemokine (C-C motif) ligand 5 (RANTES)) and reduced two anti-inflammatory cytokines (IL-4 and IL-13) as compared to the control group (Fig. 4). The cytokine responses to 50 mM dose of ethanol were compared in the presence and absence of MOR or DOR agonists (Fig. 5). Like ethanol, the MOR agonist DAMGO increased the levels of all ten pro-inflammatory cytokines and reduced the two anti-inflammatory cytokines. When combined, DAMGO was not able to change the levels of most of the pro-inflammatory and anti-inflammatory cytokines induced by ethanol, with exception of MIP-3α which was moderately elevated (Fig. 5f) and RANTES, which was moderately decreased (Fig. 5j). The DOR agonist DPDPE alone did not change the basal levels of pro-inflammatory cytokines (Fig. 5a–j) but increased the basal levels of anti-inflammatory cytokines (Fig. 5k, l). DPDPE also prevented ethanol’s stimulatory actions on pro-inflammatory cytokines and ethanol’s inhibitory actions on anti-inflammatory cytokines. These results support the concept that MOR and DOR function differently in hypothalamic microglia and identify a pro-inflammatory function for MOR and anti-inflammatory function for DOR.Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: Opioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus.

Methods: Neonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2–6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons.

Results: In the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol’s ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons.

Conclusions: These results suggest that ethanol’s neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol.

Electronic supplementary material: The online version of this article (doi:10.1186/s12974-017-0844-3) contains supplementary material, which is available to authorized users.

No MeSH data available.